INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 7, 2011

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1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 7, Ezaka.E et al., licensee IPA- Open access - Distributed under Creative Commons Attribution License 2.0 Research article ISSN Chromium (VI) tolerance of bacterial strains isolated from sewage oxidation ditch Department of Microbiology, University of Nigeria, Nsukka, Nigeria chudizoma@yahoo.com doi: /ijessi ABSTRACT The response, to different concentrations of chromium (vi), of bacterial isolates from a sewage oxidation ditch was investigated. Bacteria were isolated from sewage effluent in the oxidation ditch and chromium (vi) tolerance of the isolates determined by plating on media amended with different concentrations of the chromium. The isolates were identified as members of Staphylococcus spp., Bacillus spp., Pseudomonas spp., Micrococcus sp. and E. coli. The growth of the isolates in presence of chromium (vi) showed decrease with increasing concentrations of the metal. The inhibition was significant at chromium concentrations of 200μg/ml and above. However, the growth of the isolates in the presence of 150μg/ml chromium showed lag phases much longer than that in the absence of chromium. The isolates were resistant to high concentrations of chromium as they were able to grow at Cr (vi) concentration of up to 500 μg/ml (10 mm). As a result, the bacterial isolates obtained in this study were intrinsically resistant to high concentration of Cr (vi) and can be potential candidates in the cleanup of chromium-contaminated systems. Keywords: Chromium, tolerance, inhibition, resistance, remediation. 1. Introduction Chromium (Cr) is an important heavy metal widely used in industries. It is released into the environment by a large number of industrial operations including chrome plating, petroleum refining, leather tanning, wood preservation, textile manufacturing and pulp processing (Philip et al., 1998). Chromium exists both in hexavalent and trivalent forms. However, hexavalent form is very toxic, carcinogenic and mutagenic both in humans and animals whereas trivalent form is less toxic, less soluble and thus a lesser problem. The deposition of metallic chromium on materials imparts a refractory nature on such materials thus rendering them resistant to microbial attack and flexible over extended periods (Philip et al., 1998). More than 170,000 tone of Cr waste are discharged into the environment annually as a consequence of industrial and manufacturing activities (Kamaludeen et al., 2003). Despite the fact that heavy metals are acutely toxic to most microbes, there are heavy metal tolerant bacteria; long term exposure to metals favours proliferation of microbes that are tolerant to metals. This has been investigated by assaying habitats exposed to anthropogenic or natural metal contamination over extended period (Hutchinson et al., 1997). Heavy metals at elevated concentration are known to effect soil microbial population and their associated activities, which may directly influence the soil fertility (Smith, 1996). Microbial population has often been proposed to be an easy and sensitive indicator of anthropogenic effects on soil ecology. Cr (vi) has been reported to cause shifts in the composition of soil microbial populations, and known to cause detrimental effects on microbial cell metabolism at high concentrations (Shi et al., 2002). Received on April 2011 Published on June

2 Several physical and chemical methods exist to remove heavy metals such as chromium from the environments. However, these methods are reported to be impractical due to the operational high cost and subsequent generation of solid waste which is difficult to treat. Research in recent years indicated that many microorganisms accumulate large concentrations of metals (Ramteke, 2000). Microbial tolerance to hexavalent chromium has practical importance because it can serve as a basis for selecting organism that can be used to detoxify chromium in the environment (Ganguli and Tripathi, 2002). A number of chromium tolerant microorganisms have been reported including Pseudomonas spp. (Mondaca et al., 1998), Desulfovibrio spp. (Michel et al., 2001), Enterobacter spp (Wang et al., 1990), Escherichia coli (Shen and Wang, 1993), Bacillus spp (Camargo et al., 2003) and several other bacterial isolates (Holman et al., 1999). The objective of this study was to isolate and identify chromium (vi) tolerant bacteria from a sewage treatment plant and to evaluate their ability to tolerate different concentrations of Cr (vi). 2. Materials and Methods 2.1 Sample collection The sample site was the sewage treatment plant of the University of Nigeria, Nsukka, Southeast, Nigeria. Sewage water samples were collected with sterile containers from the oxidation ditch and transported to the laboratory immediately for analysis. 2.2 Isolation and identification of organisms The samples were serially diluted (10-fold) before plating on sterile nutrient agar plates. Some of the samples were plated on nutrient agar medium without the addition of chromium, while for the selective isolation of chromium tolerant bacteria, 50 g/ml of chromium (vi) was incorporated into nutrient agar medium. All the inoculated plates were incubated for h at 37 O C. A number of morphologically different colonies were randomly selected and sequentially subcultured for purification on the same medium. The isolates were characterized and identified using standard bacteriological techniques (Cheesborough, 2000; Holt et al., 1994). 2.3 Preparation of inoculum The isolates were, respectively, incubated overnight in nutrient broth after which the bacterial cells were harvested, washed and resuspended in distilled water. To ensure equal cell population for each of the bacterial strains, their optical density was adjusted to a uniform value using a spectrophotometer (Spec21D, Pec Medical, USA) at 600nm. 2.4 Tolerance to Cr (vi) The bacterial isolates obtained from nutrient agar with or without chromium were tested for their ability to tolerate different concentrations of Cr (vi) incorporated into nutrient broth. The effect of different concentrations of Cr (vi) on the growth of the isolates was determined by incubating the isolates in 50 ml nutrient broth contained in 250 ml Erlenmeyer flasks. The medium was amended with different concentrations of Cr (vi), namely, 50,100,150, 200 and 500 μg/ml. The flasks were incubated for 96 h during which the growth of the bacterial 1733

3 strains was determined at 24 h intervals by measuring the optical density of the cell suspension at 600 nm using a spectrophotometer (Spec21D, Pec Medical, USA). 2.5 Statistical Analysis Statistical analysis was carried out using the SPSS 13.0 for Windows software package. Data were analysed by two-way analysis of variance. Mean values were compared by least significant difference (LSD) at the 5% level using SPSS software. 3. Results To assess the possible effect of chromium stress on the isolation of the bacteria, chromium (vi) at 50 μg/ml was introduced into the isolation medium to obtain some isolates while some other isolates were obtained without chromium in the medium. Eight bacterial isolates were randomly selected, four from the medium with introduced chromium and four from medium without chromium. The four isolates from chromium-amended medium were coded CR3, CR4, CR5 and CR5B while isolates from medium without chromium were B, C7, XS, and XE. The biochemical characteristics of the isolates were determined using standard bacteriological procedures and the results are shown in table 1. Based on the results of the biochemical characteristics, the isolates were identified as members of Staphylococcus spp., Bacillus spp., Pseudomonas spp., Micrococcus sp. and E. coli. The eight selected isolates were evaluated for their ability to tolerate and grow at different concentrations (50,100,150, 200 and 500 μg/ml) of hexavalent chromium in nutrient broth medium at given time intervals. The growth of the isolates was determined by measuring the optical density of the cell suspension at 600 nm at 24 h time interval using a spectrophotometer. The results are shown in Figures 1 to 5. According to the results obtained in this study, there was no significant difference (P<0.05) in the growth of the bacterial isolates obtained with or without chromium in the isolation medium. This was shown in the growth response patterns, at all the chromium concentrations, of all the eight randomly selected isolates used in this study. In figure 1, which showed growth at chromium concentration of 50μg/ml, all the selected isolates exhibited similar growth patterns that showed slight variations that are not significant. None of the isolates showed any sign of chromium toxicity as they did not show any decrease in optical density during the study period. Isolates B and CR5B showed observable lag phase in 24 h. The growth of the isolates at a chromium concentration of 100μg/ml was evaluated and the results are shown in figure 2. All the isolates showed steady growth during the study period. Amongst the isolates, growth at 100μg/ml chromium did not show any significant difference, though for each isolate, there was significant difference in growth between the start of incubation and end of study. In figure 3, which shows the growth of the isolates at 150μg/ml, there was an extended lag period of about 48 h for almost all the isolates, except XE, which exhibited a lag period of about 24 h. However, after the lag period a steady growth increase followed till the end of the study period. At a chromium concentration of 200μg/ml, there was an indication of chromium toxicity as there was no significant difference in the growth of the isolates at the start and the 1734

4 end of the study period, except for isolate XE. This is shown in figure 4. The growth of the isolates did not show any steady increase as was observed at lower concentrations. The effect of 500μg/ml concentration of chromium (vi) on the growth of the bacterial isolates is shown in fig. 5. The analysis of the result showed that there was a sharp decline in growth within 24 hr of incubation after which the isolates showed apparent recovery. However, at the end of 96 h of incubation, none of the isolates attained a cell density as was at the beginning of the study. At 500μg/ml concentration, all the isolates attained stationary growth at 72 h after the recovery. Test Table 1: Biochemical characteristics of the isolates Isolate CR5 XS C7 CR3 R4 B CR5B XE Gram stain Motility Catalase Oxidase Indole Methylblue Malachite green Coagulase Glucose Mannitol Lactose Organism: CR5 Staphylococcus sp.; XS Staphylococcus sp. C7 Bacillus sp.; CR3 Bacillus sp. CR4 Pseudomonas sp.; B Pseudomonas sp. CR5B Micrococcus sp.; XE E. coli 1735

5 Optical density Optical density Chromium (vi) Tolerance of Bacterial Strains Isolated from Sewage Oxidation Ditch Time (h) Figure 1: Growth of the isolates in medium containing 50 g/ml of Cr (vi) Time (h) Figure 2: Growth of the isolates in medium containing 100 g/ml of Cr (vi) 1736

6 Optical density Optical density Chromium (vi) Tolerance of Bacterial Strains Isolated from Sewage Oxidation Ditch Time (h) Figure 3: Growth of the isolates in medium containing 150 g/ml of Cr(vi) Time (h) Figure 4: Growth of the isolates in medium containing 200 g/ml of Cr (vi). 1737

7 Figure 5: Growth of the isolates in medium containing 500 g/ml of Cr (vi) 4. Discussion Presence of metal tolerant bacteria in a given environment may be an indication that such area is affected by heavy metals. Such an area may foster adaptation and selection for heavy metal resistant organisms (Clausen, 2000). Isolation of bacteria from metal polluted environment would represent an appropriate practice to select metal resistant strains that could be used for heavy metal removal and bioremediation purposes (Malik, 2004). In this study, the isolation of chromium tolerant bacteria from a sewage oxidation ditch is reported. The bacterial isolates tolerated a wide range of Cr(vi) concentrations namely 50,100,150,200 and 500μg/ml. Parameswari et al. (2009) reported of bacteria resistant to up to 100mg/l Cr(vi) while chromium(vi) resistance above 2500mg/l has also been reported by Shakoori et al.(1999). In the present study, there was no significant difference in the growth responses of bacteria isolated with Cr(vi) and those isolated without chromium in the isolation medium. This might have been due to the fact that all the isolates were obtained from the same source that may have contained metal which fostered their adaptation in the presence of chromium. Hutchinson and Symington (1997) reported that long term exposure to metals imposes a selection pressure that favors the proliferation of microbes that tolerate metals. The growth response, and therefore tolerance, of the isolates was dependent on chromium concentration. The analysis of the results showed that at each chromium concentration, the isolates exhibited similar growth patterns. However, in the overall growth pattern, a decrease in growth (measured in terms of optical density) was observed upon increasing chromium concentration at any given time interval compared with the control without metal amendment. The greatest decrease was observed at the highest concentration of 500μg/ml. The inhibitive effect of Cr 6+ on the biomass growth was dependent upon the Cr 6+ concentration. Evidently, high chromate concentration prevented multiplication of bacteria (Bopp and Ehrlich, 1988). Chromate (vi) at a concentration of 500μg/ml inhibited the bacterial growth by approximately 30 to 40% with regard to control, whereas Cr 6+ at concentration of 150 μg/ml and less did not 1738

8 have significant effect on the growth of the bacterial isolates. Hassen et al. (1998) investigated the effect of heavy metals, including chromium, copper, cobalt, cadmium, zinc and mercury on Pseudomonas aeruginosa and Bacillus thuringiensis and reported that the inhibition was variable and depended on the metal and its concentrations in the medium. Chen and Wang (2007) showed that the growth inhibition of Pb 2+ on S. cerevisiae was dependent on Pb 2+ concentration. Appanna et al. (1996) reported that the effect of metal ions on the growth of Pseudomonas fluorescence depended on the metal species. Heavy metals can be toxic to microorganisms due to their strong affinity to form complexes with the cell membrane constituents, causing loss of integrity and impairment of their functions. However, microbial resistance to heavy metals is attributable to a variety of detoxifying mechanisms developed by resistant microorganisms. The heavy metal resistant organisms could be potential agents for bioremediation of heavy metal pollution. 5. Conclusion The present study revealed the capacity of bacterial isolates to tolerate and grow at different concentrations of Cr (vi). Chromium (vi) concentration can be an important environmental factor regulating tolerance to the metal. The native isolates, which tolerated high concentration of 500μg/ml of Cr (vi), can be effective in remediation strategies for ecosystem polluted with hexavalent chromium. 6. References 1. Appanna, V.D., Gazso, L.G. and Pierre M.S. (1996). Multi-metal tolerance in Pseudomonas fluorescence and its biotechnological significance. Journal of Biotechnology, 52: pp Bopp, L.H. and Ehrlich H.(1988) Chromate resistance and reduction in Pseudomonas flourescens strain LB 300. Archves of Microbiology 150: pp Camargo F.A.O., Bento F.M., Okeke B.C. and Frankenbarger W.T. (2003). Chromate reduction by chromium resistant bacteria isolated from soil ontaminated with dichromate. Journal of Environmental Quality, 32: pp Cheesbrough, M. (2000). District Laboratory Practice in Tropical Coutries. Cambridge University Press, Cambridge. 5. Chen, C. and Wang, J. (2007). Response of Saccharomyces cerevisiae to lead ion stress. Applied Microbiology and Biotechnology, 74: pp Clausen, C.A. (2000).Isolating metal-tolerant bacteria capable of removing copper, chromium, and arsenic from treated wood. Waste Management Research, 18: pp Ganguli, A. and Tripathi, A.K. (2002). Bioremediation of toxic chromium electroplating effluent by chromate reducing Pseudomonas aeruginosa A2chr in two bioreactors. Applied Microbiology and Biotechnology, 58: pp Hassen, A., Saidi, N., Cherif, M. and Boudabous, A. (1998). Effects of heavy metals on Pseudomonas aeruginosa and Bacillus thuringiensis. Bioresource Technology, 65: pp

9 9. Holman, H.Y.N., Perry D.L., Martin, M.C., Mcking, W.R. and Hunter-cwera, J.C. (1999). Real time characterization of biogeochemical reduction of Cr (vi) on basal surfaces by SR-FTIR imaging. Geomicrobiology Journal, 16: pp Holt, J.G., Krieg, R.N., Sneath, A.H.P., Staley, T.J., Williams, T.S. (1994). Bergey's Manual of Determinative Bacteriology 9th Edition. (International Edition). 11. Hutchinson,T.C. and Symington, M.S.(1997). Persistence of metal stress in a forested ecosystem near Sudbury, 66yrs after closure of the O Donnell roast bed. Journal of Geochemistry and Exploration, 58: pp Kamaludeen, S.P. B., Arukumar K.R., Avudainayagam S. and Ramasamy K. (2003). Bioremediation of chromium contaminated environments. Indian Journal of Experimental Biology, 41: pp Malik,A.(2004).Metal bioremediation through growing cells. Environ. Int. 30: pp Mondaca, M.A., Gonzalez C.L. and Zaror, C.A. (1998). Isolation, characterization and expression of a plasmid encoding chromate resistance and reduction in Pseudomonas putida KT2441. Letters in Applied Microbiology, 26: pp Michel, C. Brugma, M. Aubert, C., Bermadac. A. and Bruschi M., (2001). Enzymatic reduction of chromate: comparative studies using sulphate reducing bacteria. Applied Microbiology and Biotechnology, 55: pp Parameswari,E., Lakshmanan,A.and Thilagavathi,T.(2009). Chromate resistance and reduction by bacterial isolates. Australian Journal of Basic and Applied Science, 3: pp Philip, L., Iyengar, L. and Venkobachar, C. (1998). Cr(vi) reduction by Bacillus coagulans isolated from contaminated soils. Journal of Environmental Engineering, 124: pp Ramteke, P.W. (2000). Biosorption of nickel (ii) by Pseudomonas stutzeri. Environmental Biology, 21: pp Shakoori, AR, Maakhdoom MM and Haq, R.U. (1999). Hexavalent chromium reduction by a dichromate resistant gram-positive bacterium isolate from effluent of tanneries. 20. Appliied Micrbiology and Biotechnology, 53: pp Shen, H. and Wang Y. (1993). Characterization of enzymatic reduction of hexavalent chromium by Escherichia coli ATCC Applied and Environmental Microbiology, 59: pp Shi W, Becker J, Bischoff M, Turco RF, and Konopka AE (2002). Association of microbial community composition and activity with lead, chromium and hydrocarbon contamination. Applied and Environmental Microbiology, 68: pp

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